The objective of this work is to characterize in mechanistic detail the way by which certain hydrolytic enzymes function. Information obtained from these studies could contribute to the development of improved methods for treating and characterizing certain metabolic diseases in man which arise from an abnormality in a hydrolytic enzyme. The ionization behavior of the His-159 in papain and various inactive derivative of the enzyme will be determined using difference pmr spectroscopy, fluorescence spectroscopy and from studies of rates of chemical modification. The ionization behavior of low molecular weight amines will be studied in aqueous-organic media, in order to establish the reason for the unusual ionization behavior of the catalytically important groups at the active site of papain. The rate of cyclization of amides containing both neighboring hydroxyl and imidazolyl groups will be studied as a mode for the acylation step in the catalytic cycle of serine proteases. The effect of carboxyl, thiol, and imidazolyl modification in D-serine dehydratase will be studied, in order to identify the catalytic groups at the active site of this enzyme. A newly developed assay for plasma pyridoxal 5'-phosphate will be used to determine whether certain neurological dysfunctions in humans are associated with altered vitamin B6 metabolism.